In 2009, there were 146,970 new cases of colorectal cancer (CRC) in the U.S. resulting in 49,920 deaths (Jemal et al. CA Cancer J Clin 59 (4), 225-249 (2009); herein incorporated by reference in its entirety). The lifetime risk of an American developing CRC is ˜5.3%. Screening the asymptomatic population can prevent 65-90% of all CRCs through both diagnosis and removal of the precursor lesion, the adenomatous polyp. Unfortunately, ˜50% of the population does not undergo any screening due to concerns about cost, discomfort, complications, embarrassment, and availability. Thus, more effective screening strategies are needed. CRC screening approaches include stool, blood, radiographic and endoscopic (Lieberman, N Engl J Med 361 (12), 1179-1187 (2009); Whitlock et al. Ann Intern Med 149 (9), 638658 (2008); herein incorporated by reference in their entireties). The Multi-group Task Force recommends two classes of CRC tests: tests that target only carcinomas (e.g. stool tests) and those that are also sensitive to adenomas. The latter is strongly advocated given its potential for cancer prevention through interruption of the adenoma-carcinoma sequence (Levin et al. Gastroenterology 134 (5), 1570-1595 (2008); herein incorporated by reference in its entirety). The US Preventive Services Task Force recommends only stool tests (FOBT, not DNA), flexible sigmoidoscopy and colonoscopy (“Screening for Colorectal Cancer: U.S. Preventative Services Task Force Recommendation Statement,” Ann Intern Med (2008); herein incorporated by reference in its entirety). Flexible sigmoidoscopy (endoscopic examination of the distal colon) was a stalwart of CRC screening, but has lost favor due to inability to detect proximal neoplasia (particularly important in women) (Seeff et al. Gastroenterology 127 (6), 1670-1677 (2004); Meusink et al. Dis Colon Rectum 45 (10), 1393-1396 (2002); herein incorporated by reference in their entireties. Air-contrast barium enema is infrequently used given advent of more sensitive, less uncomfortable tests (Rockey et al. Lancet 365 (9456), 305-311 (2005); herein incorporated by reference in its entirety). Serum tests include proteomic, antibody arrays, or specific proteins (TIMP-1, CCSA-3 and CCSA-4), but lack sensitivity for advanced adenomas (Duffy et al. Eur J Cancer 39 (6), 718-727 (2003); Leman et al. Cancer Res 67 (12), 5600-5605 (2007); herein incorporated by reference in their entireties). Recent multi-center trials reported that for significant neoplasia (>10 mm), virtual colonoscopy (CT colography or CTC) had per lesion sensitivities of 84% and 80% in average and high-risk cohorts, respectively (Johnson et al. N Engl J Med 359 (12), 1207-1217 (2008); Regge et al. Jarna 301 (23), 2453-2461 (2009); herein incorporated by reference in their entireties). However, the CTC miss rate for CRCs was not trivial (˜6%). Moreover, it is impractical to refer all patients for colonoscopy for lesions identified by CTC because of cost and patient satisfaction (logistic constraints require a second visit and bowel purge for colonoscopy). However, leaving potentially premalignant lesions in place is unpalatable for most patients and physicians (Shah et al. Am J Med 122 (7), 687 e681-689 (2009); herein incorporated by reference in its entirety). Other concerns include discomfort (due to bowel purge for CTC and colonic air insufflation), radiation exposure from serial examinations, and management of extra-intestinal findings on CTC, which occur in 66% of cases with 16% deemed to require further investigation (Brenner & Georgsson, Gastroenterology 129 (1), 328-337 (2005); Kimberly et al. J Gen Intern Med 24 (1), 69-73 (2009); herein incorporated by reference in their entireties). These and other concerns led the Center for Medicare Studies to decide against reimbursing the CTC (Dhruva, et al. N Engl J Med 360 (26), 2699-2701 (2009); herein incorporated by reference in its entirety). Imaging capsule (PiliCam) has recently received considerable attention. However, this approach still needs bowel purge, is expensive, and requires a second procedure if a polyp is identified. A recent study showed poor performance for advanced adenomas (sensitivity 73% and specificity 79%) and cancers (sensitivity 74%) (Van Gossum et al. N Engl J Med 361 (3), 264-270 (2009); herein incorporated by reference in its entirety). Colonoscopy is the most accurate test (98% sensitivity for advanced adenomas) and has been demonstrated to reduce future neoplasia by an estimated 65-90% (Winawer et al. N Engl J Med 329 (27), 1977-1981 (1993); herein incorporated by reference in its entirety).
Colonoscopy will likely remain the “gold standard” of screening for the foreseeable future. The combination of diagnostic and therapeutic capabilities is particularly attractive. Unfortunately, utilizing colonoscopy for screening the entire population is impractical. There is insufficient capacity to perform colonoscopy on the entire average risk population (over 100 million Americans over age 50) (Kahi et al. Clin Gastroenterol Hepatol 7 (7), 770-775; quiz 711 (2009); Seeff et al. Gastroenterology 127 (6), 1661-1669 (2004); herein incorporated by reference in their entireties). Even if there were capacity, the cost would be prohibitive (estimates up to $50 billion per year). Complications from colonoscopy are not rare, including life-threatening issues such as bleeding or bowel perforation especially in the elderly (Rabeneck et al. Gastroenterology 135 (6), 1899-1906, 1906 e1891 (2008); Warren et al. Ann Intern Med 150 (12), 849-857, W152 (2009); herein incorporated by reference in their entireties). These limitations are juxtaposed with the remarkably low yield of screening relevant neoplasia (˜5-7%). Thus, in retrospect, more than 90% colonoscopies could possibly be deemed unnecessary.
Fecal tests for CRC screening are a minimally invasive and highly desirable option. The main advantage is excellent patient acceptability since non-compliance with invasive screening is the major problem with current CRC screening. Existing fecal tests rely on detecting consequences of tumors such as bleeding or tumor products. Indeed, the fecal occult blood test (FOBT) is widely used but is unable to detect advanced adenomas, the target of CRC screening efforts. Indeed, guaiac and even more accurate immunohistochemical techniques (Hemoccult and Hemoccult Sensa) have sensitivities between 11-21%; a stool DNA panel improved sensitivity to only 18-20% despite a marked increase in cost ($400-700 per test) (2008 study in 4,482 patients) (Imperiale et al. N Engl J Med 351 (26), 2704-2714 (2004); Ahlquist et al. Am Intern Med 149 (7), 441-450, W481 (2008); Hewitson et al. Am J Gastroenterol 103 (6), 1541-1549 (2008); herein incorporated by reference in their entireties). Thus, better fecal tests are urgently needed (Levin et al. Gastroenterology 134 (5), 1570-1595 (2008); herein incorporated by reference in its entirety).
Feces are composed of apoptotic intestinal epithelial cells (the entire lining is shed every 3-7 days), bacteria and remnants of food. There has been interest in isolating fecal colonocytes from the fecal mucus layer through immunomagnetic bead purification. However, this is expensive and cumbersome (Maisushita et al. Gastroenterology 129 (6), 1918-1927 (2005); herein incorporated by reference in its entirety). Recently, it was shown that the mucus layer of stool contains morphologically viable, non-apoptotic colonocytes. While typical fecal assays have looked for tumor cells (the “needle in a haystack” limitation), the mucus layer colonocytes are more likely to come from the normal colonocytes (abraded from the epithelium as formed stool scrapes against mucosa) (White et al. Cancer Epidemiol Biomarkers Prev 18 (7), 2006-2013 (2009); herein incorporated by reference in its entirety). Thus, the existing fecal tests are subject to the “needle in a haystack” limitation and have unacceptably poor sensitivity, especially for early curable lesions. What is needed is a fecal test for detection of CRC that is capable of early detection and does not rely on bleeding or tumor products.
A common theme in a variety of malignancies (e.g., colon, lung, head and neck, liver, etc.) is field carcinogenesis (also known as field effect, field defect or field of injury), the observation that the genetic/environmental milieu that results in colon carcinogenesis diffusely impacts upon the entire colonic mucosa (Kopelovich et al. Clin Cancer Res 5 (12), 3899-3905 (1999), Roy et al. Gastroenterology 126 (4), 1071-1081 (2004); Bernstein et al. Cancer Lett 260 (1-2), 1-10 (2008); herein incorporated by reference in their entireties). The hallmark is the clinical observation of synchronous and metachronous lesions, which frequently share similar genetic/epigenetic characteristics. (Nosho et al. Gastroenterology (2009); Konishi et al. Cancer Prev Res (Phila Pal 2 (9); 814-822 (2009); herein incorporated by reference in their entireties).
MicroRNAs (miRNAs or miRs) are small non-coding, 18-25 nucleotides long RNAs that down-regulate gene expression through binding and degrading mRNA. There has been a major interest in miRNAs and cancer. Dysregulation of ˜700 miRNAs has been implicated in carcinogenesis generally via epigenetic silencing of tumor suppressor genes/proto-oncogenes (Valeri et al. Proc Natl Acad Sci USA 107 (15), 6982-6987 (2010); herein incorporated by reference in its entirety). The role in early carcinogenesis is emphasized by the fact that ˜50% of miRNAs are located in fragile areas of the chromosome(s) (deletion/amplifications) (Slaby et al. Mol Cancer 8, 102 (2009); herein incorporated by reference in its entirety). In CRC, miRNAs modulate many critical genetic pathways (e.g. EGFR (AKT, PI-3 kinase), p53, IGF-1, COX-2, epithelial-mesenchymal transition, angiogenesis and invasion). Thus, miRNAs are critical to the entire spectrum of CRC. Given their critical role, miRNAs may serve as an early detection target (Huang et al. Int J Cancer 127 (1), 118-126 (2010); herein incorporated by reference in its entirety). Combined ROC analyses using miR-29a and miR-92a showed AUC of 0.88 and 0.77 for identifying patients with CRC vs. advanced adenomas (Hundt et al. Ann Intern Med 150 (3), 162-169 (2009); herein incorporated by reference in its entirety). There are two major pathways of colon carcinogenesis, the chromosonal instability (CIN), initiated by mutations in APC, and the mismatch repair (MMR) enzymes (most commonly hMLH and hMSH2). Importantly, both APC and MMR gene expression is regulated by miRs (miR-135 and miR-155) (Valeri et al. Proc Natl Acad Sci USA 107 (15), 6982-6987 (2010); Nagel et al. Cancer Res 68 (14), 5795-5802 (2008); herein incorporated by reference in their entireties). Data from the AOM-treated rat model suggest that miRNAs may be modulated in field carcinogenesis (Davidson et al. Carcinogenesis 30 (12), 2077-2084 (2009); herein incorporated by reference in its entirety).
While the above background most directly applies to colorectal cancer, the same considerations and implications apply to other cancers, and methods of screening. Indeed, as a biological phenomenon, field carcinogenesis has been described in multiple types of cancer.